Surfactant biocatalyst for remediation of recalcitrant organics and heavy metals

ABSTRACT

Novel strains of isolated and purified bacteria have been identified which have the ability to degrade petroleum hydrocarbons including a variety of PAHs. Several isolates also exhibit the ability to produce a biosurfactant. The combination of the biosurfactant-producing ability along with the ability to degrade PAHs enhances the efficiency with which PAHs may be degraded. Additionally, the biosurfactant also provides an additional ability to bind heavy metal ions for removal from a soil or aquatic environment.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Contract No.DE-AC09-96SR18500 awarded by the United States Department of Energy. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention is directed towards bacterial strains useful forbioremediation and processes for using the bacterial strains. Inparticular, it relates to unique bacterial isolates that can degradepolyaromatic hydrocarbons (PAHs) and methods to use these novelbacterial strains for bioremediation.

BACKGROUND OF THE INVENTION

Various scientific articles and patents are referred to throughout thespecification. These publications are incorporated herein by referenceto describe the state of the art to which this invention pertains and toprovide details on standard methodologies and apparatuses which may beuseful in practicing various embodiments of the present invention.

Polyaromatic hydrocarbons (PAHs) are widespread, common pollutantsparticularly found in association with oil refineries, certain refinedpetroleum products, petroleum storage locations, and petroleum spillsites. High levels of PAHs are associated with mutagenic andcarcinogenic effects in humans and pose a high risk for migration to andpollution of soil and ground water sources. As a result, there has beenconsiderable interest in techniques and processes which will degradePAHs and related petroleum products to remediate the environment. Theuses of biological agents to treat PAHs are well known within the art.U.S. Pat. No. 6,503,746 to Daane describes bacterial strains in thefamily Bacillaceae which are used in PAH remediation efforts.

U.S. Pat. No. 3,616,204 to Linn discloses inoculating contaminated soilwith cultures of microorganisms known to degrade the unwantedcontaminants. The procedure described in Linn additionally involvesintroducing nutritional supplements to increase the soil remediationefficiency.

U.S. Pat. No. 5,100,455 to Pickard discloses using indigenous microfloraand fauna in combination with humic substrates to biologically treatsoil contaminants including petroleum associated hydrocarbons.

U.S. Pat. No. 4,849,360 to Norris discloses a bioreactor for treatingpetroleum contaminated soil in which air is forced through thecontaminated soil to facilitate the bioremediation. The bioreactor usesindigenous microflora which are supplemented with phosphorus andnitrogen nutrients.

While a variety of PAH-degrading bacteria are known and have beenutilized in various applications for remediation, there remains a needfor improvement in the art in terms of identifying new and usefulspecies having novel properties which are effective for the rapiddegradation of petroleum pollutants.

SUMMARY OF THE INVENTION

The present invention relates to methods for the degradation ofpetroleum pollutants including polyaromatic hydrocarbons (PAHs).Additionally, the present invention relates to a biotreatment processwhich enhances the removal of heavy metals from soil. The presentinvention uses isolated and purified bacterial strains of bacterialisolates from an oil refinery field. Certain of the isolates having afurther ability to produce useful biosurfactants.

It is one aspect of at least one of the present embodiments of thepresent invention to provide isolated bacterial strains that producebiosurfactants under in situ and ex situ remediation conditions. Theinnate ability of the isolated and purified bacterial strains to producebiosurfactants contributes to the remediation properties of thebacteria. The biosurfactant provides increased solubility of PAHs andaccess of the bacteria to the PAHs, thereby increasing the efficiency ofthe bioremediation by the bacteria strains.

An additional aspect of at least one of the embodiments of the presentinvention is related to isolated and purified strains of bacteria inwhich the surfactant producing properties contribute to enhancedsolubilization of petroleum and petroleum-derived products. Thebiosurfactants increase the solubilization of the petroleum productswhich promotes the aqueous flushing or removal of petroleum productsassociated with biosurfactant aggregates such as micelles and relatedstructures. Further, the biosurfactants also increase thebioavailability of petroleum products that enhance the microbial abilityto degrade contaminants. The enhanced bioavailability is beneficial tothe isolated and purified strains as well as other beneficialmicroorganisms present in the contaminated substrate.

It is yet another aspect of at least one of the embodiments of thepresent invention to provide for strains of isolated and purifiedbacteria which degrade 2- to 3-ringed low molecular weight PAHs such asnaphthalene, phenanthrene, and fluoranthene along with PAH degradationintermediates.

It is yet a further aspect of at least one of the embodiments of thepresent invention to provide for a strain of isolated, purified bacteriawhich degrades 4-ring and higher molecular weight PAHs including pyreneand fluoranthene. Typically, the 4-ring and higher PAHs are much morepersistent in the environment and resistant to degradation compared tolow molecular weight PAHs. Accordingly, the ability to provide strainsof bacteria which degrade 4-ring and higher high molecular weight PAHsis significant.

The PAH degradation intermediates may further function as metalchelators. This chelating activity or metal complexation may assistremediation in waste containing both PAHs and metals. Additionally, atleast some of the bacterial strains identified herein have an ability todegrade several different types of PAHs (including 2-, 3-, and 4-ringPAHs) in addition to the ability to degrade phenanthrene.

It is yet another aspect of at least one of the embodiments of thepresent invention to provide strains of isolated and purified bacteriahaving surfactant properties useful in the removal of metals fromcontaminated soil and substrates. The isolated and purified strains ofbacteria produce biosurfactant monomers. The biosurfactant monomers areproduced in sufficient quantity that the monomers aggregate intothree-dimensional structures including micelles. The biosurfactantmicelles define polar head groups which bind with metal ions in thesoil. The micelles, containing the metal ions, can be removed by aqueoussuspensions or flushing, thereby lowering the metal ion content of thesubstrate. The resulting removed metals, contained within thebiosurfactant micelles, are then more easily separated and concentratedfor efficient disposal or storage.

It is yet another aspect of at least one of the present embodiments ofthe invention to provide for isolated and purified cultures of bacteriawhich produce biosurfactants under constitutive conditions, the isolatedstrains being further able to degrade PAHs during bioremediationconditions.

It is yet another aspect of at least one of the present embodiments ofthe invention to provide for isolated and purified strains of bacteriahaving the ability to degrade a broad range of different types of PAHsunder bioremediation conditions.

It is yet another aspect at least one of the present embodiments of theinvention to provide for isolated and purified strains of bacteriahaving an ability to bring about a general reduction of total petroleumhydrocarbons (TPH). In addition, the ability of certain of the isolatesto produce a biosurfactant during bioremediation conditions increasesthe bioavailability of petroleum hydrocarbons to other microorganismsthat may be present within the contaminated soil or other waste product.

It is yet another aspect of at least one of the present embodiments ofthe invention to provide for isolated and purified strains of bacteriahaving the ability to degrade PAHs along with an ability to produce abiosurfactant during bioremediation conditions.

These and other aspects of the invention are provided by biologicallypure bacterial strains for bioremediation of petroleum products and PAHscomprising isolates selected from the isolates identified in Table 2.

Other aspects of at least one embodiment of this invention include aprocess of bioremediation of petroleum pollutants from a contaminatedenvironment comprising the steps of providing a supply of a substratecontaminated with a petroleum pollutant; introducing into the supply ofcontaminated substrate at least one bacteria isolate which metabolizesconstituents of the petroleum pollutant and which further produces abiosurfactant; and, providing adequate nutrients for a treatment timesufficient for the petroleum pollutant utilizing isolate to degrade thepetroleum pollution to a target concentration of 100 ppm TPH or less.

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims.

DESCRIPTION OF PREFERRED EMBODIMENT

A fully and enabling disclosure of the present invention, including thebest mode thereof, to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification. Reference nowwill be made in detail to the embodiments of the invention, one or moreexamples of which are set forth below. Each example is provided by wayof explanation of the invention, not limitation of the invention. Infact, it will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Forinstance, features described as part of one embodiment can be used onanother embodiment to yield a still further embodiment. Thus, it isintended that the present invention cover such modifications andvariations as come within the scope of the appended claims and theirequivalents. Other objects, features, and aspects of the presentinvention are disclosed in the following detailed description. It is tobe understood by one of ordinary skill in the art that the presentdiscussion is a description of exemplary embodiments only and is notintended as limiting the broader aspects of the present invention, whichbroader aspects are embodied in the exemplary constructions.

The present invention is directed to bacterial isolates obtained from acentury-old Czechowice oil refinery in Poland. The aged sludge from theoil refinery is characterized by its acidic (pH 2) properties andcontains high concentrations of PAHs along with heavy metals.Additionally, the sludge is characterized by the presence of spentcatalysts, asphaltics, diatomaceous earth, silica gel, and coal fly ash,all containing high background levels of heavy metals (Pb/Cd/Zn) whichhave been previously deposited at the site. The collection site is froman area having approximately 120,000 tons of waste material deposited inunlined lagoons 3 meters deep covering an area of 3.8 hectares. A totalof 45 bacteria, 68 fungi, and 7 yeast species were isolated from thesludge on an acidic minimum medium (pH 4) exposed to naphthalene vapor.

A subset of isolates was characterized by traditional taxonomiccriteria, BIOLOG™, and analysis of SSU rRNA genes. The bacterial groupsincluded Proteo bacteria, Ralstonia, Pseudomonas, and Alcaligenesspecies. Further characterizations of the isolates may be seen inreference to the information provided in Tables 1 and 2. The BIOLOG™characterization protocols using minimal nutritional factors along withvarious organic substrates of interest are described in reference to thepublication Use of BIOLOG™ Technology for Hazardous Chemical Screening,Microbiological Techniques 18: 329-347, 1993, and which is incorporatedherein by reference.

A total of 45 bacteria, 68 fungi, and 7 yeast species were isolatedusing a naphthalene vapor acidic mineral salts basal growth medium.While not separately set forth, it is noted that many of the isolateshave the ability to metabolize catechol and the bacterial isolates werecharacterized by the ability to degrade PAHs. Additionally, it is notedthat the isolated and purified organisms having the ability to degradePAHs also have the ability to degrade a variety of petroleum pollutantsassociated with measurements of total petroleum hydrocarbons.

As set forth below in Table 1, three bacterial species designatedCZORL1B, BP20, and CZORL1Bsm, and which correspond to isolates 1 through3 in Table 2, were observed to produce a surfactant when grown in aminimal medium containing naphthalene, phenanthrene, or fluoranthene.Nine additional strains identified in Table 1 were observed to degrade arange of PAHs indicating the isolates have a catalytic or enzymaticability to degrade the contaminants although the additional isolates donot demonstrate an ability to produce a surfactant.

The above identified bacterial strains are grown and maintained on 1percent peptone, trypticase, yeast extract, glucose (PTYG) plates. Thebacteria were grown aerobically at 30° C. and maintained on a minimalmedium at 4° C. or long-term storage in a frozen medium maintained at−70° C. or in liquid nitrogen (−196° C.).

The identification of the bacteria was made using rDNA or Fatty AcidMethyl Esters (FAME) identification protocols as set forth in thepublication Bacterial Evolution, Microbial Reviews 51: 221-271 by C. R.Woese (1987) and which is incorporated herein by reference. Deposits ofisolates 1 through 12 as identified in Table 2 were deposited with theAmerican Type Culture Collection (ATCC), Rockville, Md., on Oct. 9,2003, and have the indicated ATCC designation numbers. The deposit formsaccompanying each of the isolate deposits as submitted to the ATCC areincorporated herein by reference.

TABLE 1 Dihydroxylating dioxygenase and 2,3 catechol dioxygenaseactivity and PAH degradative range of isolates. Assayed for thefollowing characteristics:^(a) Isolate indigo meta fission NAP PHE ANTFLE ACE FLA PYR Isolated on naphthalene vapor^(b) BAA-1 (PB19) indigoBAA (PB16) indigo BP19A(PB17) indigo BAB (PB14) indigo CZORL1B metafission (KN-1)^(c) BP20 CZORL1B meta fission (KN-2)^(c) CZORL1Bsm metafission (KN-3)^(c) PB15 indigo NAP (was designated BP20) Isolated withphenanthrene overspray^(d) BPA indigo PHE ANT FLE BPB indigo PHE ANT FLEBPC indigo PHE ANT FLE BPD indigo PHE ANT FLE BPE indigo PHE ANT FLE BPFindigo meta fission NAP PHE ANT FLE ACE FLA PYR BPG indigo meta fissionBPH indigo meta fission PHE ANT FLE FLA PYR BPI indigo PHE ANT FLE BPJindigo ANT FLE BPK PHE ANT FLE BPL indigo PHE FLE BPM indigo PHE ANT FLEPYR BPN indigo PHE ANT FLE FLA BPO indigo PHE ANT FLE BPP indigo PHE BPQindigo PHE ANT FLE BPR indigo meta fission PHE ^(a)Abbreviations;indigo, production of indigo from indole; meta fission, 2,3 catecholdioxygenase activity; NAP, naphthalene; PHE, phenanthrene; ANT,anthracene; FLE, fluorene; ACE, acenanphthene; FLA, fluoranthene; PYR,pyrene. ^(b)Bacteria isolated on agar plates exposed to naphthalene.^(c)Bacteria produced biosurfactant ^(d)Bacteria isolated on agar platesexposed to phenanthrene crystals.

TABLE 2 Isolate identification. ATCC Accession Isolate IdentificationNumber 1) CZOR-L1B ALCALIGENES-PIECHAUDII SRS PTA-5580 (KN-1) 2) BP-20(KN-2) RALSTONIA PICKETTII SRS. PTA-5579 3) CZOR-L1BsmPSEUDOMONAS-PUTIDA BIOTYPE B PTA-5581 (KN-3) SRS 4) BPB FLEXIBACTER CF.SANCTI SRS PTA-5570 5) BPC PSEUDOMONAS FREDRIKSBERGENSIS PTA-5571 SRS 6)BPE STAPHYLOCOCCUS WARNERI. LMG PTA-5572 19417 SRS 7) BPF SPHINGOMONASSRS PTA-5573 8) BPH SPHINGOMONAS SP. S37 SRS PTA-5574 9) BPIPHYLOBACTERIUM SRS PTA-5575 (α PROTEOBACTERIUM TA-A1) 10) BPJ SERRATIAFICARIA SRS PTA-5576 (α PROTEOBACTERIUM TA12-21) 11) BPK AGROBACTERIUMTUMEFACIENS SRS PTA-5577 12) BPL RHIZOBIUM SP. SDW045 SRS PTA-5578

The above identified bacteria isolates have been established as distinctspecies. Each of the identified isolates has PAH-degrading propertiesand have demonstrated an ability to reduce TPH in soil as well. Inaddition, certain isolates have the ability to produce a biosurfactant.Each isolate is believed novel, based upon the rDNA characterization andvariations noted in Tables 1 and 2 with respect to physiological growthcharacteristics.

The isolates 1-3, ATCC PTA-5580 (Alcaligenes-piechaudii SRS); ATCCPTA-5579, (Ralstonia pickettii SRS); and ATCC PTA-5581(Psuedomonas-putida Biotype B SRS) identified above, all demonstrate theability to produce a biosurfactant, the formation of which was notedduring culturing conditions. The biosurfactant exudate was evaluated foreach isolate and determined to have a surface tension altering propertyconsistent with a surfactant. Isolates 4-12 all demonstrate the abilityto biodegrade a variety of PAHs (Table 1). As set forth in Example 1below, the use of a consortium of the twelve isolates identified inTable 2 to remediate petroleum hydrocarbons contained in soil in abioreactor remediation study results in visible quantities ofbiosurfactants being produced under the bioremediation conditions.

The ability of certain of the isolates to produce bioreactants isbelieved to enhance remediation through several different mechanisms.The production of the biosurfactant increases the biologicalavailability of PAHs and other hydrophobic petroleum compounds. Theincreased biological availability includes the ability of the producedsurfactant to solubilize and make available to the isolate the PAHs andother petroleum compounds. As such, the isolates' ability to producesurfactants increases the efficiency of the isolates to degrade andmetabolize PAHs.

As noted in Example 1, the consortium of isolates used results invisible quantities of surfactants being produced within the soil.Biosurfactants are generally known to have a chemistry consisting of apolar head and a non-polar tail. In aqueous solutions, biosurfactantsserve to reduce liquid surface tension and to facilitate the formationof an emulsion between liquids of different polarities. This abilityfacilitates the biosurfactants' usefulness in that hydrophobic,non-polar tail regions of the biosurfactants and biosurfactant micellesmay trap oils and other petroleum compounds. The trapped oils andpetroleum compounds have greater bioavailability to bacteria forbiodegradation. Additionally, micelles containing trapped oils andpetroleum compounds may be periodically removed or flushed from thesystem, thereby providing an ability to further isolate and separatepetroleum compounds from the soil substrate.

Additionally, micelles formed by the biosurfactants promote the removalof metals from the soil. The hydrophilic polar head groups of micelleswill bind metal and metal ions present within the soil. Once bound, thesoluble nature of the micelles allows the micelles and bound metals tobe collected. Once collected, the now concentrated volume of micellesand contained metals can be further treated to separate the metals fromthe biosurfactant.

EXAMPLE 1

A mobile bioreactor was constructed and was supplied with a four tonvolume of soil contaminated with low level cesium-137 and 26,000 ppmpetroleum hydrocarbons. The contaminated soil was weathered materialobtained from the Savannah River Site (Aiken, S.C.). The source and makeup of the petroleum products is unknown but believed to be a mixture ofused motor oil and diesel fuel.

The soil was amended with a 7% bulking agent of aged compost. For eachisolate, a three liter culture in log growth phase was added anddistributed within the four tons of mixed waste soil. The bioreactor isequipped with a raised, secondary, perforated floor having bottom feedaeration lines which provide a continuous supply of ambient air to thebioreactor. Additionally, periodic nutrient supplements of nitrogen,potassium, and phosphorus fertilizers (10-10-10) were applied to enhancethe biological activity within the bioreactor. Influent and effluentwater couplings were attached. Air compressors, vacuum pumps, and aliquid pump were used to control and regulate the air and liquid flowsthrough the bioreactor and control moisture content in the bioreactor.

The presence of low level cesium-137 limited the number and types ofsampling techniques used to monitor the bioreactor and required the useof HEPA filtering with the air effluent couplings. Periodic CO₂measurements indicated that, in a five-month interval, 121 pounds ofpetroleum products were degraded. Based upon the CO₂ measurements, it isconservatively estimated that the bioremediation process removed 16,000mg/kg of petroleum contaminants from the soil during the five-monthevaluation interval. It is believed that within a 14-month timeinterval, the four ton volume of contaminated soil will have the TPHreduced to a level less than about 100 ppm based upon an averagebiodegradation of 62 gm/day observed over the course of the samplinginterval.

The ability of the isolates to degrade petroleum and other hydrocarbonproducts while producing biosurfactants offers enormous advantages interms of efficiency and versatility of treatment protocols. Forinstance, it is believed that for soils contaminated solely withpetroleum and petroleum by-products, the present isolates may, eitherindividually or as a consortium, be used with conventionalbioremediation techniques to improve the efficiency of degradation. Asdescribed above, the action of the biosurfactants creates a greater zoneof petroleum solubility for each individual bacterium. As a result, agreater availability of petroleum products occurs. Further, to theextent the isolates form aggregate colonies, biofilms, or biosheetswithin portions of the soil, the surfactants are believed tosubstantially increase the bioavailability of the petroleum substratesfor the bacterial aggregates. At the same time, the surfactants alsoincrease the solubilization of heavy metals that may be present andprovide an ability to reduce the heavy metal concentration by removal ofthe produced surfactants.

As seen in Example 1, the consortium of isolates provides for abioremediation process which can achieve significant reductions inpetroleum from contaminated soil. This property is particularly usefulwith respect to formulating disposal strategies for mixed waste in whichpetroleum contaminated soil and low-level radioactive material arepresent together. Currently, soil contaminated with low-levelradioactive waste and having additional petroleum contaminants must bebelow regulatory limits of 1 ppm for BTEX (benzene, toluene,ethylbenzene, xylene, and 100 ppm TPH (Total Petroleum Hydrocarbons)before the soil can be classified and disposed of as a low-levelradioactive waste. The cost of disposing of soil which meets thedefinition of a low-level radioactive waste is approximately $262 percubic meter per year. In contrast, soil containing both low-levelradioactive material and petroleum contamination in excess of theregulatory limits must be stored as a mixed waste product. The cost ofstorage of mixed waste soil is approximately $10,165 per cubic meter ofsoil per year based on yearly costs alone. The ability to treat mixedwaste soils and thereby remove substantial levels of petroleumcontaminants is of critical importance. Removing sufficient petroleumcontaminants from a mixed waste soil allows the waste to be disposed ofas a low-level radioactive waste. The resulting cost is 38 times lowerthan the storage cost of a mixed waste.

The use of the present inoculants is further advantageous in that,unlike some prior art techniques, the volume of amendments to the soilis kept at a minimum. Keeping the volume of soil amendments to a minimumreduces the eventual disposal costs, particularly for soil containinglow-level radiation.

The present isolates are also believed useful for in situ remediationprojects. The consortium of isolates may be supplied to contaminatedsoil using any number of conventional techniques. As needed, nutritionalsupplements along with the supply of oxygen in either a physical orchemical form facilitates the bioremediation activity. Given theisolates' ability to degrade PAHs as well as the desired ability todegrade petroleum hydrocarbons generally, in situ remediation using theisolates is advantageous. Additionally, the ability of certain of theisolates to produce a surfactant (biosurfactant) during soil growthconditions makes the use of certain isolates more beneficial. As noted,the biosurfactant enhances the ability to physically entrap petroleumproducts and heavy metals as well as providing for increasedsolubilization and access of petroleum hydrocarbons to both thebacterial isolates as well as native microorganisms present within thesoil environment.

Although preferred embodiments of the invention have been describedusing specific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the art withoutdeparting from the spirit or the scope of the present invention, whichis set forth in the following claims. In addition, it should beunderstood that aspects of the various embodiments may be interchanged,both in whole and in part. Therefore, the spirit and scope of theappended claims should not be limited to the description of thepreferred versions contained therein.

1. A biologically pure bacterial strain for bioremediation of petroleumhydrocarbons, said strain selected from the group consisting of ATCCaccession numbers Flexibacter cf. sancti srs, PTA-5570; Pseudomonasfredriksbergensis srs, PTA-5571; Staphylococcus warneri. Img 19417 srs,PTA-5572; Sphingomonas srs, PTA-5573; Sphingomonas sp. s37 srs,PTA-5574; Phylobacterium srs, PTA-5575; Serratia ficaria srs, PTA-5576;Agrobacterium tumefaciens srs, PTA-5577; Rhizobium sp. sdw045 srs,PTA-5578; Ralstonia pickettii srs, PTA-5579; Alcaligenes-piechaudii srs,PTA-5580; and Pseudomonas-putida biotype b srs, PTA-5581.
 2. Abiologically pure strain and mutants thereof, having all of theidentifying characteristics of said strain, wherein said strain iscapable of degrading petroleum hydrocarbons, said strain being selectedfrom the group consisting of ATCC accession numbers Flexibacter cf.sancti srs, PTA-5570; Pseudomonas fredriksbergensis srs, PTA-5571;Staphylococcus warneri. Imp 19417 srs, PTA-5572; Sphingomonas srs,PTA-5573; Sphingomonas sp. s37 srs, PTA-5574; Phylobacterium srs,PTA-5575; Serratia ficaria srs, PTA-5576; Agrobacterium tumefaciens srs,PTA-5577; Rhizobium sp. sdw045 srs, PTA-5578; Ralstonia pickettii srs,PTA-5579; Alcaligenes-piechaudii srs, PTA-5580; and Pseudomonas-putidabiotype b srs, PTA-5581.
 3. A biologically pure strain according toclaim 2 wherein said hydrocarbon is a polyaromatic hydrocarbon.